Gel electrophoresis is commonly used in molecular biology to separate molecular components of a sample based on size and charge. Classically these components are transferred onto a membrane. Transfer procedures or blotting include fluid flow as in Southern blotting or electroblotting as in U.S. Pat. No. 4,589,965. The product, termed a blot, is treated to make the bands of separated molecules detectable visually or by some other detection means. For example, autoradiographic detection is used where the treatment includes radioactive labeling. Fluorescent markers, activated by a UV source, are used and detected by a two-dimensional charge coupled device as described in U.S. Pat. No. 4,874,492.
Chemiluminescence via enzymatic triggering of certain substituted 1,2-dioxetanes is a preferred method of creating a readily detectable signal. Various aspects of this art are disclosed in a number of patents.
U.S. Pat. No. 4,857,652 to Schapp discloses light producing 1,2-dioxetanes of the formula ##STR1## wherein ArOX is an aryl ring substituted with an X oxy group and A are passive organic groups which allow the 1,2-dioxetane to produce light when triggered by removing X. X is a chemically labile group which is removed by an activating agent. The 1,2-dioxetane compounds can be triggered to produce light at room temperatures.
U.S. Pat. No. 4,952,707 to Edwards et al., affords a general description of enzymatically-cleavable 1,2-dioxetanes. This patent describes enzymatically-cleavable chemiluminescent 1,2-dioxetanes having the formula: ##STR2## wherein R.sub.1 is hydrogen, or a bond when R.sub.2 is a substituent bound to the dioxetane ring through a spiro linkage, or an organic substituent that does not interfere with the production of light; R.sub.2 is a fused polycyclic ring-containing fluorophore moiety having an enzymatically-cleavable, labile ring substituent; and T is a stabilizing group that prevents the dioxetane compound from decomposing before the enzymatically-cleavable labile ring substituent's bond is cleaved.
U.S. Pat. No. 4,956,477 to Bronstein et al., also describes the synthesis of enzyme-cleavable 1,2-dioxetanes, useful for chemiluminescent immunoassays, DNA probe assays, and direct assays for an enzyme.
U.S. Pat. No. 4,959,182 to Schaap describes a method and composition for providing enhanced chemiluminescence from 1,2-dioxetanes. In this method an enzyme cleavable 1,2-dioxetane is mixed with a surfactant and a fluorescent compound attached to a hydrocarbon to form a co-surfactant in a micelle or other structure. This method provides an enhancement of 500 fold in signal for enzyme-triggered chemiluminescence of 1,2-dioxetanes in solution. Moreover, Bronstein et al., (J. Biolumin. Chemilumin. 4, 99-111, 1989) report that bovine serum albumin and other water-soluble macromolecules provide a significant enhancement of chemiluminescent signal generated from enzyme-cleavable 1,2-dioxetanes in solution. All of these enhancers are believed to increase the stability of the anion intermediate and the light-emitting species by keeping them in a hydrophobic environment. However, on a hydrophobic support such as a nylon membrane, the support provides a hydrophobic environment for the anionic species. Consequently, no significant enhancement is provided by these enhancers when the enzyme is immobilized on a hydrophobic support.
AU-A36340/89 to Okada et al. describes a method for enhancing the chemiluminescent signal from enzyme-triggered 1,2-dioxetanes. The enzymatic reaction is performed at the optimum pH for the enzyme. Afterwards the pH is increased by the addition of strong base to enhance the luminescent reaction. Increases in signal from 7 to 59 fold were reported for assays done on polystyrene beads.
WO89/06650 to Bronstein et al. discusses dioxetanes for use in assays, and including a fluorescent chromophore spiro-bound at the 4-carbon of the dioxetane. The dioxetane has the formula: ##STR3## where X is CR.sub.7 R.sub.8, O, S, or R-R (where each R.sub.7, R.sub.8, and R, independently, is H, alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, cycloheteroalkyl, aralkyl, aklaryl, or an enzyme cleavable group). Each R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6, independently, is H, an electron withdrawing group, an electron donating group, heteroaryl, or an enzyme cleavable group, or groups R.sub.1 -R.sub.6 together form a ring. T is a substituted or unsubstituted aryl, polyaryl, cycloalkylidene or polycycloaklylidene group spiro-bound at the 3-carbon of the dioxetane. These dioxetanes are used in an assay to detect a member of a specific binding pair or an enzyme.
U.S. Pat. No. 4,705,847 to Hummelen et al. relates to a process for preparing substituted polycyclo-alkylidene polycyclo-alkanes, such as substituted adamantylidene adamantanes, and the corresponding epidioxy compounds. The polycycloalkylidene polycycloalkanes are halogenated, and thereafter the halogenation product is optionally subjected to a substitution reaction. The resulting products are converted to the corresponding epidioxy compounds. Various epodioxy compounds are disclosed which contain a dioxetane ring. These compounds are useful as thermochemiluminescent labels.
U.S. Pat. No. 4,948,975 to Erwin et al., describes a quantitative luminescence imaging system which provides a means to measure low light levels from luminescent reactions in electromagnetic fields, e.g., microwave radiation, and its use in the areas of chemiluminescent assays and thermal microdosimetry. The effect of the microwave radiation on chemiluminescence described in the patent and in a publication (Kiel, Bioelectromagnetics 4, 193-204, 1983) is significantly different from the instant description. The system described involves enzymatic reactions, specifically the oxidation of luminol catalyzed by peroxidase enzymes, in protein gels which are kept wet with solution. The enhancement affected by microwave radiation is due to an increased mobility of substrate (hydrogen peroxide) within the gel.
Method and apparatus for handling and processing blot membranes during blotting, hybridization and detection is disclosed in U.S. Pat. No. 4,812,216 and related U.S. Pat. No. 4,913,791. These references disclose a method and apparatus for supporting and handling blot membranes during the course of blotting, analysis, and storage. An apparatus is disclosed including membrane support means in combination with one or more receptacles in a cooperating, releasable lock or key means so that the membrane is received in the same orientation from receptacle to receptacle. A method is also disclosed including providing a membrane and support means suitable secured to one another (such as by interlocking or adhesive) and whereby the membrane is handled conveniently by the support means without disturbing the membrane or biological material bound thereto. The membrane is transported as a single piece part arrangement to and from processing stations. However, these references do not teach an automated and controlled assembly for processing of a membrane including a biological sample, wherein the membrane is desirably contacted by a pressure head and enclosed within light shielding apparatus during the detection stage.
U.S. Pat. No. 30,595 discloses a container for dispensing reagent slides into apparatus which carry out quantitative chemical analysis of fluid samples. A generally rectangular housing includes a chamber for receiving a stack of the slides. The container fits into a complementary shaped nest in the analysis apparatus, and discontinuity means are included to inhibit insertion at an improper orientation. U.S. Pat. No. 4,152,390 discloses a chemical analyzer comprising a plurality of cartridges containing test slides. A slide transfer mechanism feed slides from a cartridge, transports them to a metering device for fluid deposit, and delivers the slides by conveyor means through an incubator and to an analysis means. However, these references do not teach the combination of guide apparatus to position a transfer membrane, a pressure head for releasable engagement, and light shielding apparatus which selectively encloses about the membrane as in the present invention.
It is an object of the present invention to provide apparatus for handling electrophoretic transfer membranes which provides and controls: means for receiving a transfer membrane and locating that membrane for detection, automatically controlled actuation of the detection means, means for membrane discharge, and means for resetting so that a subsequent transfer membrane may be processed. It is a further object of the present invention to provide apparatus useful in chemiluminescent detection wherein the chemiluminescent signal is enhanced. A feature of the present invention is the incorporation of a pressure head within the apparatus which distorts the membrane and optionally heats the membrane desirably for improved signal strength. It is an advantage of the present invention that the apparatus and the process disclosed herein enable accurate and controlled processing of electrophoretic samples with minimal involvement of skilled laboratorians. These and other objects, features, and advantages of the present invention will become apparent upon having reference to the following description of the invention.